Therapeutic vaccine for treatment of diabetes type 1 in children, application of the cell sorter and the method of multiplying treg cells to produce therapeutic vaccine for treatment of diabetes type 1

ABSTRACT

The gist of the invention consists in the therapeutic vaccine for treatment of diabetes type 1 in children, which contains
         Treg cells CD3(+)CD4(+)CD25(high)CD127(−). Claimed too is the cell sorter used to produce the vaccine and the method of multiplying Treg cells in vitro.

FIELD OF THE INVENTION

The present invention concerns therapeutic vaccine for treatment of diabetes type 1 in children, application of the cell sorter to produce therapeutic vaccine for treatment of diabetes type 1, and the method of multiplying Treg cells in vitro to produce therapeutic vaccine for treatment of diabetes type 1.

PRIOR ART

Diabetes type 1 (DM1A) is a genetically linked disease, however all researchers agree that direct damage to β cells in the pancreas is due to autoimmune reaction. Speaking in favour thereof are both the presence of antibodies towards β cell antigens, and lymphocytic infiltration to the islets of Langerhans, or the so-called insulitis, accompanied by increased β cell apoptosis.

The regulatory T-lymphocytes (Treg) form a specific population in the immune system. Although accounting for less than 1% of the leucocytes in the peripheral blood, they regulate the immune response so that swift elimination of harmful pathogens is possible while our own tissues remain protected. This is because Treg cells do not block other cells of the immune system when foreign pathogens are attacked, but prove strongly inhibiting when the immune system begins to destroy our own tissues and organs. Therefore by analogy, the immunosuppressive action of Treg cells sometimes earns them the name of the “intelligent steroids”.

Reduced numbers of Treg cells in the organism are associated with transplant failures and incidence of allergic and autoimmune diseases. One of the diseases characterised by numerical deficiency of Treg cells, is diabetes type 1 where autoimmune attack destroys the patient's pancreas.

Known from the international publication WO 2004/110373 is a vaccine composition that comprises modified insulin B chain components suitable for use as immunogenic agents for treatment and prevention of type 1 diabetes.

Known from publication WO 2012/001099, on the other hand, is vaccine containing at least one enterovirus selected from the group including: Coxsackie viruses CAV4, CAV5, CAVE, and echovirus E18, or its component. The description states that the listed enteroviruses are linked to diabetes type 1, which opens new therapeutic and diagnostic possibilities.

Similarly, the description of WO 2012/001100 discloses vaccine comprising e.g. Coxsackie B virus CBV1 to prevent or treat diabetes type 1. It has been found that the virus is strongly associated with the risk of contracting diabetes type 1.

The above publications disclose vaccines intended for treatment of diabetes type 1, nevertheless they are different than the vaccine being the gist of this invention.

In order to increase the effectiveness of treating diabetes type 1 in children it is necessary to search for more effective and successful methods of treating the disease.

GIST OF THE INVENTION

Unexpectedly, it has been found that the new therapeutic vaccine for treatment of diabetes type 1 prevents the disease effectively. Administration of the vaccine according to this invention to patients results in an increase in the primary marker of the pancreas function, i.e. the C-peptide level. In addition, the sorter employed was originally dedicated to cell therapies, which enhances its safety.

The gist of the invention consists in the therapeutic vaccine for treatment of diabetes type 1 in children, which contains:

-   -   Treg cells CD3(+)CD4(+)CD25(high)CD127(−).

The gist of this invention further consists in employing the cell sorter to produce the therapeutic vaccine for treatment of diabetes type 1, where the cells are sorted to isolate Treg cells using the algorithm sorting out the following phenotype:

-   -   CD3(+)CD4(+)CD25(high)CD127(−)doublet(−)lineage(−)dead(−).

The gist of this invention also consists in the method of multiplying Treg cells in vitro to produce therapeutic vaccine for treatment of diabetes type 1, where:

-   -   lymphocytes T CD4+ are isolated by the immunomagnetic method and         marked with monoclonal antibodies,     -   the sorter-isolated Treg cells are multiplied in the CellGro or         X-VIVO medium supplemented with autological inactivated serum         and interleukin-2,     -   the culture is supplemented with artificial antigen-presenting         cells in the 1:1 proportion.

Selected for the marking are the following monoclonal antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD14, anti-CD16, anti-CD25, and anti-CD127.

The monoclonal antibodies CD3, CD4, CD8, CD19, CD14, CD16, CD25, and CD127 recognise the antigens, and are conjugated with fluorescent dyes.

The antigen-presenting artificial cells are magnetic beads coated with anti-CD3 and anti-CD28 antibodies.

THE FIGURES

FIG. 1—presents the level of Treg cells CD3+CD4+CD25highCD127-FoxP3+ in children suffering from diabetes type 1, subject to the Treg cell therapy (n=10) over four months' observation. The value at point “−10 days” represents the day the blood was drawn for Treg cell isolation. The grey columns present the results obtained for children not administered the Treg lymphocyte infusion (control group; n=10). The values are given at their median, minimum, and the maximum levels. The statistically significant values (p<0.05) are marked with “*”.

FIG. 2—presents the C-peptide, the daily insulin dose/kg BW(DDI/kg), and the HbA1C in the tested children with diabetes type 1, subject to the Treg cells therapy (n=10) over the four months' observation. The results for the patients not administered the cell preparation (control group; n=10) are presented in grey columns. The values are given at their median, minimum, and the maximum levels, and the statistically significant values (p<0.05) are marked with “*”.

The invention is illustrated with the following embodiment, which is exemplary, i.e. not limiting in nature.

EXEMPLARY EMBODIMENT

250 ml of peripheral blood was sampled from each patient with the assistance from an anaesthesiologist. In the case of children whose body weight was less than 50 kg the sampled blood volume accounted for 0.5% of the body weight (BW). This concerns patients under the age of 18.

The collected blood was processed at the Regional Centre of Blood Donation and Treatment in Gdansk to extract the buffy coat and serum. Isolated from the buffy coat were peripheral blood mononuclear cells (PBMC) through centrifuging in the Ficoll/Uropolin concentration gradient. Lymphocytes T CD4+ were then separated by the immunomagnetic method (separation purity: 96-99%) using the CD4+T enrichment kit and marked with the following monoclonal antibodies (mAb): anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD14, anti-CD16, anti-CD25, and anti-CD127 (5 ul mAb/10⁶ cells). Among the listed antibodies those which recognize antigens CD14, CD16, CD19, and CD8 were conjugated with the same dye. The purpose of that dying scheme was to exclude the cells positive with respect to the listed antigens (i.e. monocytes, NK cells, lymphocytes B and cytotoxic T lymphocytes) without the need to introduce additional fluorochromes, which reduces the undesirable phenomenon of fluorescent spectra overlapping. Then, the cells were sorted to separate Tregs using a sorting cytometer to the algorithm sorting the following phenotype: CD3(+)CD4(+)CD25(high)CD127(−)doublet(−)lineage(−)dead(−).

The adopted exemplary dying scheme (antibody; dye name acronym, full name of the dye)

-   -   antiCD127 FITC (Fluorescein isothiocyanate)     -   antiCD25 PE (phycoerythrin)     -   antiCD16 PerCP (Peridinin Chlorophyll Protein Complex)     -   antiCD19 PerCP (Peridinin Chlorophyll Protein Complex)     -   antiCD8 PerCP (Peridinin Chlorophyll Protein Complex)     -   antiCD14 PerCP (Peridinin Chlorophyll Protein Complex)     -   antiCD4 APC (allophycocyanin)     -   antiCD3 Pacific Blue/Pacific Blue         or equivalents evoked to emit fluorescent light in similar         spectrum ranges.

The purity of the thus isolated Treg cells was ˜100% [median(min-max): 98%(97-99)]. An important modification compared to our earlier procedure consisted in applying the Influx cell sorter designed in accordance with the good manufacturing practices (GMP). The sorter is fitted with a replaceable sample flow line, which eliminates the risk of sample cross-contamination among the patients. Moreover, applied was the CellGro medium meeting the GMP standards or X-VIVO. The medium was supplemented with autological inactivated serum (10%) and interleukin-2 (1000 U/ml). Introduced into the culture were the so-called antigen-presenting artificial cells [magnetic beads coated with anti-CD3 and anti-CD28 antibodies in the 1:1 proportion. The cells were cultivated until the appropriate number was attained, though no longer than for 2 weeks [median(min-max): 10 days (7-12)].

The above indicated modifications allowed the attainment of substantially improved stability and quality of the cultured Treg cells in the final product. The actual application of the preparation in therapy was conditional on satisfaction of the following criteria: factor FoxP3 expression above 90% [median(min-max)=93%(90-97)], positive result of the IFNγ production inhibition test, and negative results of microbiological tests—no genetic material of the HBV, HCV, or HIV viruses, and no bacterial contamination in the culture supernatants. Before infusion, the cells were washed with PBS, the magnetic beads removed, and administered in slow intravenous injection in 250 ml 0.9% NaCl under supervision of the anaesthesiologist within 1 h after the product release. The therapeutic dose was 20×10⁶/kg BW (n=6), or 10×10⁶/kg BW (n=4; whenever no higher number of cells had been achieved upon cultivation for 2 weeks), or 30×10⁶/kg body weight. The control group was made up of patients who met all above-listed criteria of inclusion in the test, except for appropriate venous access, hence were not treated with the Treg vaccine. The test was not randomised, nor was there a blank sample introduced, and the children of the control group were not subject to any medical intervention related to the pending tests (blood sampling, simulated transfusion, or the like). Table 1 provides the characteristics of the tested groups. The test endpoints were as follows: the fasting C-peptide level, the HbA1c concentration, the insulin requirement, especially the daily dose (DDI)=0.5 UI/kg BW adopted as the remission indicator. The test was conducted in accordance with the procedure approved by the Independent Research Bioethics Committee at the Medical University of Gdansk (NKEBN/8/2010). A written consent to the above procedure was obtained from each patient and the parents.

None of the patients was observed to develop any serious infections, episodes of acute hyper-/hypoglycaemia, or any other undesirable side effects of the Tregs vaccine at any time over the test period. In case of one patient the Treg cell infusion date coincided with flu diagnosed a day after the Treg cells had been administered.

Beginning on the infusion date and continuously afterwards the recorded Treg lymphocyte percent level in the peripheral blood was significantly increased (Wilcoxon test, p=0.04) (FIG. 1).

Two weeks after the Treg cell infusion all patients subject to the therapy were observed to demonstrate substantially reduced demand for exogenous insulin and a reduced HbA1c level (FIG. 2).

The first significant differences between the test group and the patients of the control group were observed six months after formulation of the diabetes diagnosis (5-6 months after the Treg cell infusion). The treated patients continued in the remission phase [DDI median(min-max)=0.24 UI/kg BW (0-0.55)], whereas the control group experienced the end of remission [DDI median(min-max)=0.55 UI/kg BW (0.43-0.69)] (Mann-Whitney U test, p=0.03). In addition, the children treated with Treg cells proved to have a significantly higher level of C-peptide [median(min-max): 0.65 ng/ml (0.46-2.11) vs. 0.40 ng/ml (0.15-0.54)] (Mann-Whitney U test, p=0.04) (FIG. 3). No differences with respect to therapy effectiveness were observed in the patients who had been administered Treg cells dosed at 20×10⁶/kg BW or 10×10⁶/kg BW. Therefore, all results of the test group are presented en block.

LITERATURE

-   1. Marek N, Krzystyniak A, Ergenc I, Cochet O, Misawa R, Wang L J,     Gołąb K, Wang X, Kilimnik G, Hara M, Kizilel S, Trzonkowski P,     Millis J M, Witkowski P. Coating human pancreatic islets with     CD4(+)CD25(high)CD127(−) regulatory T cells as a novel approach for     the local immunoprotection. Ann Surg. 2011; 254(3):512-8; discussion     518-9. -   2. Marek N, Bieniaszewska M, Krzystyniak A, Juscinska J, Mysliwska     J, Witkowski P, Hellmann A, Trzonkowski P. The time is crucial for     exvivo expansion of T regulatory cells for therapy. Cell Transplant.     2011 (20):1747-1758; -   3. Trzonkowski P. All roads lead to T regulatory cells.     Transplantation. 2011; 91(2):150-1. -   4. Trzonkowski P, Bieniaszewska M, Juścińska J, Dobyszuk A,     Krzystyniak A, Marek N, Myśliwska J, Hellmann A. First-in-man     clinical results of the treatment of patients with graft versus host     disease with human ex vivo expanded CD4+CD25+CD127− T regulatory     cells. Clin Immunol. 2009; 133(1):22-6. -   5. Trzonkowski P, Szaryńska M, Myśliwska J, Myśliwski A. Ex vivo     expansion of CD4(+)CD25(+) T regulatory cells for immunosuppressive     therapy. Cytometry A. 2009; 75(3):175-88. -   6. Ryba M, Marek N, Hak Ł, Rybarczyk-Kapturska K, Myśliwiec M,     Trzonkowski P, Myśliwska J. Anti-TNF rescue CD4+Foxp3+ regulatory T     cells in patients with type 1 diabetes from effects mediated by TNF.     Cytokine. 2011; 55(3):353-61. -   7. Trzonkowski P, Szmit E, Myśliwska J, Myśliwski A. CD4+CD25+T     regulatory cells inhibit cytotoxic activity of CTL and NK cells in     humans-impact of immunosenescence. Clin Immunol. 2006;     119(3):307-16. -   8. Trzonkowski P, Szmit E, Myśliwska J, Dobyszuk A, Myśliwski A.     CD4+CD25+T regulatory cells inhibit cytotoxic activity of T CD8+ and     NK lymphocytes in the direct cell-to-cell interaction. Clin Immunol.     2004; 112(3):258-67. -   9. Trzonkowski P, Zaucha J M, Mysliwska J, Balon J, Szmit E,     Halaburda K, Bieniaszewska M, Mlotkowska M, Hellmann A, Mysliwski A.     Differences in kinetics of donor lymphoid cells in response to G-CSF     administration may affect the incidence and severity of acute GvHD     in respective HLA-identical sibling recipients. Med Oncol. 2004;     21(1):81-94. -   10. Gołąb K, Krzystyniak A, Marek-Trzonkowska N, Misawa R, Wang L J,     Wang X, Cochet O, Tibudan M, Langa P, Millis J M, Trzonkowski P.,     Witkowski P. Impact of culture medium on CD4+CD25highCD127lo/neg     Treg expansion for the purpose of clinical application. Int     Immunopharmacol. 2013. doi:pii: S1567-5769(13)00058-1.     10.1016/j.intimp.2013.02.016

TABLE 1 CLINICAL CHARACTERISTICS OF THE PATIENTS Untreated Treg treated control group (n = 10) (n = 10) Age (years) [median; min-max] 12.2; 8-16   11.8; 7-16   BMI [median; min-max] 16.9; 14.2-21.5 16.9; 14.2-20.7 Glycaemia on empty stomach at 354; 151-588  354; 151-598  diagnosis (mg %) median; min-max] Polydipsia at diagnosis 5 8 (number of patients) Polyuria at diagnosis 5 8 (number of patients) Body weight loss at diagnosis 4 3 (number of patients) Capillary blood pH at diagnosis 7.39; 7.36-7.46 7.39; 7.34-7.53 [median; min-max] Capillary blood pO₂ at 69.3; 63.4-88.0 69.0; 56.9-86.2 diagnosis (mmHg) [median; min-max] Capillary blood pCO₂ at 39.1; 28.0-41.8 38.0; 24.0-41.0 diagnosis (mmHg) [median; min-max] HCO₃ at diagnosis 23.85; 18.8-25.0  23.3; 21.3-25.2 (capillary blood - mmHg) [median; min-max] Acid base balance (BE - mEq/l) −0.55; −7.8-1.0  −0.6; −7.0-0.9  [median; min-max] Capillary blood Sat02 at diagnosis 94.1; 90.2-97.3 95.5; 92.4-98.0 (%) [median; min-max] Ig anti-GAD65 (number of patients) 10  10  Ig anti-ICA (number of patients) 5 5 Ig anti-IAA (number of patients) 9 8 

1-6. (canceled)
 7. A method of producing a population of cells comprising CD3(+)CD4(+)CD25(high)CD127(−) Treg cells, the method comprising: sorting a population of cells using a cell sorter to isolate cells having the phenotype CD3 (+)CD4(+)CD25 (high)CD 127(−] doublet(−)lineage(−)dead(−).
 8. The method of claim 7, wherein the cell sorter comprises a replaceable sample flow line.
 9. The method of claim 7, wherein the step of sorting comprises excluding cells that are positive for antigens selected from the group consisting of: CD14, CD16, CD19, CD8, and combinations thereof.
 10. A method of multiplying CD3(+)CD4(+)CD25(high)CD127(−) Treg cells in vitro comprising: isolating CD4⁺ T lymphocytes using an immunomagnetic method; marking the CD4⁺ T lymphocytes with one or more monoclonal antibodies; sorting the CD4+T lymphocytes using a cell sorter to isolate cells having the phenotype CD3(+)CD4(+)CD25(high)CD127(−]doublet(−)lineage(−)dead(−) to generate a population of sorted Treg cells; multiplying the sorted Treg cells in CellGro or X-VIVO medium supplemented with autologous inactivate serum and interleukin-2; and adding artificial antigen-presenting cells to the medium in a 1:1 proportion to the sorted Treg cells.
 11. The method of claim 10, wherein the step of marking comprises marking with monoclonal antibodies selected from the group consisting of: an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD19 antibody, an anti-CD14 antibody, an anti-CD16 antibody, an anti-CD25 antibody, an anti-CD127 antibody, and combinations thereof.
 12. The method of claim 10, wherein the one or more monoclonal antibodies are conjugated to fluorescent dyes.
 13. The method of claim 10, wherein the artificial antigen-presenting cells comprise magnetic beads coated with anti-CD3 and anti-CD28 antibodies.
 14. The method of claim 10, wherein the CellGro or X-VIVO culture medium is supplemented with at least 1000 U/ml interleukin-2 and approximately 10% autologous inactivated serum.
 15. The method of claim 10, wherein the sorted Treg cells comprise a Treg cell population having a purity of at least 97%.
 16. The method of claim 10, wherein the sorted Treg cells are propagated for a time that does not exceed 2 weeks.
 17. The method of claim 10, wherein the artificial antigen-presenting cells are in the shape of a magnetic sphere.
 18. The method of claim 10, wherein the multiplied Treg cells are tested for a FoxP3 expression level above 90%, a positive result in an IFN-gamma inhibition assay, and a negative result in a microbiological assay. 